In order to study the codon- anticodon interaction of seleno-tRNAGlu from Clostridium sticklandii, glutamate codons GAA and GAG were synthesized. Under near physiological conditions, seleno-tRNAGlu interacts almost equally well with both codons, while Escherichia coli tRNA2Glu, which contains 5-mnm2-s-U instead of 5-mnm-2-se-U at the wobble position, showed a profound preference of GAA over GAG. Similar results were observed at lower pH (PH 5.5). Deselenization of seleno-tRNAGlu abolished the codon stimulated ribosome binding activity. In a cell free protein translation system [3H] Glu-seleno-tRNAGlu is a very active substrate for the incoprporation of glutamate into the nascent protein. Genomic DNA fragments of C. sticklandii (approximately 4 K bp), which contained the seleno-tRNAGlu gene were ligated to plasmid puc 8 and used to transform E. coli cells (strain HB 101). Two positive clones have been found. This serves as an effective starting point for further work at the genetic level. Expression of the cloned seleno-tRNA Glu gene may produce significant amount of tRNA precursors which can be modified with selenium. The presence of selenium-containing tRNAs in various rat tissue (heart, kidney, testis and liver) was investigated. Bulk tRNAs prepared from liver contanied the highest amount of selenium (about 0.6%). Many different selenium-containing species which can be resolved on a HPLC DEAE column were present in the preparations. Although the selenium component(s) are alkaline labile, they are not seleno amino acids because no selenomthione or selenocysteine was detected. The amount of seleno-tRNAs in the total tRNA populations of a cultured plant cell line, Daucas carota, ranged from 0.54% to 1.2%. Total nucleoside analysis of bulk tRNA indicated that the selenium-containing component(s) were very hydrophobic in character.